Figure 4. Effect of LY75 knockdown on SKOV3 cell proliferation migration and invasion.

A. Cell proliferation of LY75 knockdown clones sh-S3 and sh-S6 was compared to the control clone (Ctrl); B. Western blot analysis of the proliferation marker Ki-67 in LY75 knockdown clones sh-S3 and sh-S6 compared to the control clone. C. Cell migration of LY75 knockdown clones sh-S3 and sh-S6 was compared to the control clone (Ctrl). Migration was assessed using Boyden-chamber assay. Cells from the LY75 knockdown clones sh-S3 and sh-S6 and the Ctrl clone were seeded into the upper chambers in 0.1% FBS containing medium at a density of 2.5 × 10⁴ per well, and 600 μl of 1% FBS containing medium was placed in the lower chamber as a chemoattractant. After 24 h at 37°C in 5% CO₂, the cells were fixed with cold methanol and stained with blue trypan solution. Migrated cells on the underside of the filter were photographed and counted by phase contrast microscopy. E. Cell invasion was assayed in a similar way, as the upper chambers were coated with Matrigel. Here, NIH3T3 conditioned medium was added in the lower chamber as a chemoattractant (see Methods for details). All experiments were performed in triplicate. For each experiment, cell number was calculated as the total count from 10 random fields per filter (at magnification × 40). The bar graphs in panels D and F. represent quantitative determinations of migration and invasion data obtained by selecting 10 random fields per filter under phase contrast microscopy and results are expressed as % change of the sh-S3 and sh-S6 clones over the Ctrl clone. Differences between shRNA-LY75 transfected and vehicle- transfected SKOV3 cells were determined by a Student's t-test; error bars denote mean ± SEM; *indicates statistical significance (p < 0.05).